Kinesin-2 motors carry out anterograde transport in cilia and flagella, as well as other bidirectional transport processes in cells. Although many aspects of kinesin mechanochemistry are understood, the specific motor activities and regulation that underlie multi-motor and bidirectional cargo transport are not well understood. Deficiencies in Kinesin-2 transport lead to abnormal development, photoreceptor degradation and polycystic kidney disease. The goals of this project are to understand the mechanism by which Kinesin-2 motors walk along microtubules, and the degree to which Kinesin-2 motor properties are tuned for its specific transport tasks. A notable difference from the canonical Kinesin-1 motor family is that neck linker domain of Kinesin-2, which connects the core motor head to the coiled-coil domain, is 17 amino acids compared to only 14 in Kinesin-1. Because the neck linker serves as a mechanical element connecting each head to their shared coil-coil, it is expected that extending the neck linker will diminish mechanochemical coupling between the head domains. Consistent with this, we found that extending the Kinesin-1 neck linker diminishes its processivity and shortening the Kinesin-2 neck linker enhances processivity. In addition, we found that the force dependence of Kinesin-2 velocity and run length differ from Kinesin-1, suggesting the motor may be optimally tuned for bidirectional cargo transport rather than long distance unidirectional transport. Using single-molecule and multi-motor experiments in conjunction with computational modeling of the kinesin chemomechanical cycle, we will uncover the structural basis of mechanistic differences between Kinesin-1 and Kinesin-2 motors, with the goal of understanding bidirectional transport in vivo. This work involves structural studies to determine the role played by the neck linker and neck coil domains in processive movement along microtubules. We will also investigate specific steps in the Kinesin-2 chemomechanical cycle to determine how the motor biochemistry is controlled by intramolecular tension between the two head domains, as well as by external loads applied by optical tweezers. These investigations into the mechanism of Kinesin-2 motility will provide a framework in which to understand Kinesin-2 transport in cells. By benchmarking against Kinesin-1, these measurements will establish universal themes underlying kinesin mechanochemistry that will help to better understand the molecular basis of neurodegenerative diseases and aid the effort to develop anti-tumor therapies targeting mitotic kinesins.